Antisense RNAs and DNAs have proved to be effective agents for selectively inhibiting certain genetic sequences in cell-free systems as well as within the living cell. Their mode of activity is based on the specific recognition of a complementary nucleic acid strand and attachment thereto, thus affecting the transcription, translation and cleaving processes. This mechanism of activity theoretically makes it possible to use antisense oligonucleotides as therapeutic agents which will block the expression of certain genes (such as deregulated oncogenes or viral genes) in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells and perform their inhibiting activity therein (Zamecnik et al., 1986), even though the intracellular concentration thereof is low, partly because of their restricted uptake through the cell membrane owing to the strong negative charge of the nucleic acids.
Another method of selectively inhibiting genes consists in the application of ribozymes, i.e. RNA molecules which recognise specific RNA sequences and are able to bind to them and cleave them. Here again there is the need to guarantee the highest possible concentration of active ribozymes in the cell, for which transportation into the cell is one of the limiting factors.
In order to counteract this limiting factor, a number of solutions have already been proposed.
One of these solutions consists in direct modification of nucleic acids, e.g. by substituting the charged phosphodiester groups with uncharged methyl phosphonates (Smith et al., 1986), carbamates (Stirchak et al., 1987) or silyl compounds (Cormier et al., 1988) or using phosphorothioates (Stern et al., 1988). Another possible method of direct modification consists in the use of nucleoside analogues (Morvan et al., 1988, Praseuth et al., 1988)).
Even though some of these proposals appear to offer a promising way of solving the problem, they do have numerous disadvantages, e.g. reduced binding to the target molecule and a reduced inhibitory effect. A chief disadvantage of the in vivo use of modified oligonucleotides is the possible toxicity of these compounds.
An alternative method to the direct modification of the oligonucleotides consists in leaving the oligonucleotide itself unchanged and providing it with a group which will impart the desired properties to it, e.g. with molecules which will make transportation into the cell easier. One of the proposed solutions within the scope of this principle consists in conjugating the oligonucleotide with polycationic compounds (Lemaitre et al., 1987).
Various techniques are known for genetic transformation of mammalian cells in vitro, but the use of these techniques in vivo is restricted (these include the introduction of DNA by means of viruses, liposomes, electroporation, microinjection, cell fusion, DEAE dextran or the calcium phosphate precipitation method). the
Attempts have therefore already been made to develop a soluble system which can be used in vivo, which will convey DNA into the cells in a directed manner by means of receptor-mediated endocytosis (G. Y. Wu, C. H. Wu, 1987). This system was developed for hepatocytes and is based essentially on the following two facts:
1. On the surface of the hepatocytes there are receptors which bind specific glycoproteins and convey them into the cell. PA0 2. DNA can be bound to polycationic compounds, e.g. polylysine, by a strong electrostatic interaction, forming soluble complexes.
The system is based on the principle of coupling polylysine with a glycoprotein of a kind to which the receptor will respond and then, by adding DNA, forming a soluble glycoprotein/polylysine/DNA complex which will be transported to the cells containing the glycoprotein receptor and, after the absorption of the complex into the cell, will make it possible for the DNA sequence to be expressed.